Phenotypic mixing of host range and serological specificities in bacteriophages T2 and T4

VIROLOGY

2,

388-398 (1956)

Phenotypic Mixing of Host Range Specificities in Bacteriophages GEORGE California

Institute

and Serological T2 and T4l

STREISINGER~

of Technology,

Accepted February

Pasadena,

California3

25, 1966

Crosses between h2+ (T2 host range) and h4+ (T4 host range) phage produce progeny which are phenotypically h 2+, h4+, or hz+h4+. The proportions of genetically h4+ and h2+ particles belonging to any one phenotypic class are similar. The host range and serological specificities which are controlled by one genetic locus are also linked phenotypically. It is suggested that t.he materials responsible for these specificities are produced in a pool and become randomly associated with the genetic material of phage during maturation. INTRODUCTION

Crosses between the related phages T2 and T4 produce some progeny particles in which the phenotype does not correspond to the genotype (Delbriick and Bailey, 1946). These unusual particles were found by Novick and Szilard (1951) to be phenotypically like T4, in that they had the T4 host range, but to be genetically T2, in that their progeny, after one or more multiplication cycles, had the T2 host range. It was further shown by Delbriick (personal communication) that the unusual T2 was neutralized by both anti-T2 and anti-T4 sera at similar rates as T2 and T4 respectively. Hershey and Rotman (1949) found that progeny of T2 h+ X T2 h crosses produced particles genetically T2 h, but phenotypically h+. The process producing these part,icles was named “phenotypic mixing” (Hershey et al., 1951). Hershey further observed (personal communi1 This work was presented in partial fulfillment of the requirements for the Ph.D. degree, University of Illinois, Urbana, and was supported in part by a grant from the American Cancer Society (on recommendation of the Committee on Growth) to Dr. S. E. Luria. 2 This work was carried out while the author was a Public Health Service Research Fellow of the National Institutes of Health at the University of Illinois, Urbana. 3 Present address: Carnegie Institution, Cold Spring Harbor, N. Y. 388

PHENOTYPIC

MIXING

OF

BACTERIOPHAGE

389

cation) that in crosses of T2 rh X T2 r+h+ similar proportions of the r+h recombinant particles and of the rh progeny particles had h+ phenotypes. The differences in host range between T2 and T4 are determined by t,he allelic loci h*+ and h4+ respectively (Streisinger, 1956). Studies of phenotypic mixing were undertaken, utilizing crosses between hackcrossed stocks that were isogenic for most loci except the host range and an r marker locus. The results reported here suggest that the host range phenotypes are associated randomly with the genotypes. MATERIALS

AND

METHODS

The materials and met,hods employed have been described in the preceding paper (Streisinger, 1956). All crosses involve the backcross stocks described in that paper. EXPERIMENTAL

Assortment of host range phenotype with genotype. Progeny phage particles from crosses h4+ X h*+ car1 be classified with respect to host range phenotype by measuring their adsorbability on the indicators B/2 and B/4. Particles with h4+ phenotype will adsorb on B/2, while particles wit,h IL?+ phenotype will adsorb on B/4. Classification wit,h respect t,o host range genotype can be accomplished by scoring clear and turbid plaque types on mixed indicators B+B/4 (Streisinger, 1956). The various genotypic classes of progeny phage from crosses rp2h4+ X r+h?+ and r+h4+ X r2,h2+ were analyzed for their phenotypes by measuring the fractions of phage adsorbable on B,l4 and on B/2 (Table 1). The phage particles fall into three classes with respect, to phenotype: h”+ like (adsorbable on B/2), h*+ like (adsorbable on Bji) and a third class which is both like h4+ and like h*+ (adsorbable on both B/2 and B,i4). ,4 possible fourth class, adsorbable on the common host B, but on neither Bj4 nor B/2 was smaller than 3% of the total phage. Note t)hat each phenotype is distributed among all genotypes. Control lysates of each of the parental phages were prepared urlder conditions identical to those used for the crosses and subsequently mixed. Over 95% of the h*+ phage of such mixtures was adsorbed by B,‘4, and none by B/2; over 95 % of the h4+ of the mixtures was adsorbed by B/2, but none by B/4. Progeny phage from the crosses, when adsorbed by B under conditions of single infection and t,hen permitted to undergo a single cycle of growt,h,

TABLE ADSORBABILITY

Cross number and cross

1. hb+rB x h2+rf

4. h4+r+ x hzfm

OF

PHAGE

adsorbable on B/2 adsorbable on B/4 adsorbable on B/2 and B/4 absorbable on B/2 only adsorbable on B/4 only

1 h2+ X h4+

FROM

CROSSES

Frequencies of phenotypic each of the genotypic

Properties of fraction

adsorbable on B/2 adsorbable on B/4 adsorbable on B/2 and B/4 adsorbable on B/2 only adsorbable on B/4 only

PROGENY

class in classes

Phzm&ypic ha+r+

hz+m

h’+r+

h*+ and h4+h2+

89%

85%

‘33%

64%

h2+ and h4+h2’

28%

27%

48%

62%

h4fh2t

17%

12%

16%

26%

h4+

72%

73%

52%

38%

h2+

11%

15%

32%

36%

h4+ and h4+h2+

80%

8’3%

79%

82%

h2+ and h4+hzf

51%

53%

61%

57%

h4thZt

33%

41%

42%

40%

h4f

47%

45%

37%

40%

h2+

18%

12%

19%

15%

PROCEDURE: Lysates from crosses were mixed with young cells of B/2 or B/4 in the presence of 1OP M sodium cyanide. The adsorption time was sufficient to assure adsorption of at least 95yo of the adsorbable phage. The cells were removed by low speed centrifugation and the supernatants were scored for genotypes by platings on B+B/4. From the genotype frequencies in the unadsorbed and in the adsorbed samples, the frequencies of the various phenotypic classes in each genotype class were calculated. The fractions adsorbable on both B/2 and B/4 were calculated by subtracting 1 from the sum of the fractions adsorbable on B/2 plus the fractions adsorbable on B/4. The fractions adsorbable only on B/2 (or B/4) were calculated by subtracting the fractions adsorbable on both B/2 and B/4 from the fractions adsorbable on B/2 (or B/4). The tests with lysate from cross 4 were carried out in triplicate; the values are mean percentages. The values for cross 4 are corrected for a fraction nonadsorbable on a mixture of B/2 and B/4. This fraction was less than 3% of the total.

390

PHENOTYPIC

produced phage which tjwo parental types. Thus it is clear that may have phenot(ypes t,ypes whose genotypic able t,o adsorb to both The degree

MIXI?rrG

OF

391

BACTERIOPHAGE

behaved identically

to artificial

mixtures of the

progeny phage particles from a cross P+ X P differing from their genotypes and even phenocounterpart is not observed (sucahas t.hc partklcs B/2 and B/-C).

qf phenotypic

mixing

at various times during

the latent period.

In order to determine whether the association between phenot’ype and genot,ype changed during the course of the latent period an aliquot, of the infect’ed cells of cross r+h4+ X r2zhz+ was lysed prematurely (Doermann, 1952); another aliquot was permitted t)o lyse at the normal time, while in a third aliyuot lysis was delayed (Doermann, 1948). As shown in Table 2, t,he fraction of particles adsorbable on both B/2 and on H;4 increased, but the proportions of the various genotypes in eac+hphellotypic fraction remained approximately t,he same. Th,e fate of phenotypically mixed phage after adsorption,. The experimerits described above est’ablish that some phage of each genotype c~l11tl TABLE

2

PHENOTYPES OF PHAGE PROGENY FROM CROSS h4+r+ X hZCrzz (CROSS 4) AFTER VARIOUS TIMES OF INTRACELLULAR DEVELOPMB~ST

Time of lysis

15 minutes 21 lninutes 360 minutes

Frequency of recombinants Phtszypic between m and h4+

__-.

10%

12%

h2+

32%

15 minutes 21 minutes 360 minutes

h4+

15 minutes 21 minut,es 360 minutes

h2+h4+

Fequencies of phenotypic class in each of the genotypic classes

~~-.

h%s

h’-,+

!I%

18%

14%

12%

19%

15%

24%

16%

67% 45% 45%

4!,7* 37%

65%

21%

3ooj, +a% 49yo

h%t

h%+

12% 18% 12%

9%

49% 47%

32% 37% 33% 53%

41%

4270

24%

40% 37% 15%

42% 45%

KOTE: Sodium cyanide lo-* M was added to the infected bacteria 15 minutes after the initiation of development to obtain premature Iysis. An aliquot, of the infecting mixture, at a multiplicity of 10, was added to the infected bn&eria t,o inhibit, lysis (Uoermann, 1948). Ammonium su1fat.e was added to a 5oj, conrentration at the time of lysis t,o prevent, readsorption of liberated phage (French el ul., 1952). The phenotypir classes and genotypic classes were determined as descbribed in Table 1.

392

GEORGE

STREISINGER

TABLE ADSORPTION

AND IXFECTING

ABILITY

3

OF PHAGE

(CROSS

FROM CROSS h4+rz2 X hz+r+

1) Frequencies of genotypes

Treatment

Adsorption B/4

Adsorption

B/2

Mode of recovery

on

on

infective centers (from platings on B+B/4 after serum inactivation of unadsorbed phage) unadsorbed phage (supernatant plated on B+B/4 after centrifugation) sum of infective centers plus unadsorbed phage infective centers (from platings on B+B/4 after serum inactivation of unadsorbed phage) unadsorbed phage (supernatant plated on B+B/4 after centrifugation) sum of infective centers plus unadsorbed phage

hl+m

h’+r+

h”fl23

29%

44%

43%

71%

73%

52%

28%

100%

117%

95%

86%

he++

89%

92%

63%

54%

11%

15%

31%

36%

100%

107%

94%

90%

attach to B/2 or B/4. Further experiments were designed to measure the ability of adsorbed particles to produce progeny in the bacteria they were adsorbed to. Mixed phage from a cross h2+r+ X h4+r22were adsorbed to B/4 and to B/2 (single infection). The fractions of progeny particles of each genotype that were left unadsorbed as well as the fractions which adsorbed and produced progeny were measured (Table 3). All of the phage originally present was recovered in one or the other of these two fractions, showing that practically all adsorbed phage particles proceeded to multiply. This result was confirmed by a one-step growth experiment of the same mixed phage on B/4. The mean burst size of the adsorbed phage of all genotypes was about the same as that of a control h2+ stock, and the bursts were produced after a normal latent period. The production of both phenotypes in mixedly injected bacteria. In order t,o determine whether each bacterium mixedly infected with h2+ and h4+ particles produced progeny particles with h2+ as well as h4+ phenotypes the yields from single bacteria were examined. B cells were mixedly

PHENOTYPIC

MIXING

OF

TABLE

393

BACTERIOPHSGE

4

LIBERATION OF h2+ AND h4+ PHENOTYPES FROM CELLS OF STRAIN B INFECTED WITH h2+ AND ha+ PARTICLES Input multiplicities __-~~ .~~-~ h2+ h’+ parent parent

6.5 I3 12 11 4

5 IO 14 1.x 16

h2+ G=;z;;Pe

i&z+ Phenotype

plaques on B+B/Q)

93 96 114 92 85

90 84 85 102 52

hsf Phenotype among those liberating h2+ genotype

97 88 74 110 61

h’* G”$WF piiques on B+B/2)

90 101) 106 92 99

h”? Phenotype kkiques on B/2)

101 83 104 81 106

MIXEULP

h4Phenotype among those liberating hr+ genotype

116 83 98 8X 107

NOTE: Mixtures of phage at various multiplicities were added to starved B cells suspended in buffer. After a period for adsorption unadsorbed phage particles were removed by antiserum, and the cells were plated on various indicators or mixtures of indicators. The plaque counts (= infective centers) were compared \vit h those on B t,aken as 100%.

infected with h2+ and h4+ particles either at equal or at unequal multiplicities. The infected cells were plated before lysis on B+B/3 and on R/4 as well as on B+B/2 and B/2 (Table 4). At the multiplicities used almost all of the infected bacteria yield phage particles of each genotype. Half of the yield of a bacterium will consist,, for instance, of genetically I?+ particles. It should be recalled that all genetically h’+ part,icles will give clear plaques on B+B/4 but that only those genetically I?+ particles which in addition are also phenotypically I?+, will give plaques on B/4. Bacteria liberating phage particles of exclusively one phenotype, for inst,ance h4+, will produce no plaques on B/4. Thus, the plaques on B/‘4 measure the number of infective centers in which V+ phenotype is produced. This number is compared with the number of bacteria liberating genetically I?+ particles (clear plaques on B+B:4). The same procedure using the indicators B/2 and B+B/2 measures the fract,ion of bacteria in which U+ phenotype is produced. This procedure (aan give only a minimal estimate of the number of bacteria liberating phage part,icles with either phenot,ype. A mixedly infected cell liberating only one genetically I?+ particle, for instance, will be scored as a clear plaque on B+Bj4. This one particle might be phenotypically U+, and not, be scored on B/4 even though other, phenotypically V+ particles may br present in the same cell. The resulk, shown in Table 4, indicate that! wit,h equal input of t,he

394

GEORGE

STREISINGER

TABLE SERUM

NEUTRALIZATION

OF PROGENY

5

FROM CROSS h4+r+

x

h2+r22 (CROSS

4)

Fractions of phage neutralized Serum

anti-T4 (a) anti-T2 (b) inactivable by both anti-T4 and antiT2 sera (c) (c = a + b - 100%)

h”+m

h”r+

hz+m

h%+

94%

73% 56% 29%

52% 69%

‘31% 61% 22%

63% 27%

21%

NOTE: The serum tests were carried out in duplicate using two different anti-T2 sera and two different anti-T4 sera. The figures given are the means for each pair of sera. The heterologous activity of these sera had been removed by absorption of the serum with heterologous phage. The serum treatment was sufficient to neutralize over 95% of control homologous phage (inactivation of phage from the cross may not have proceeded to completion, hence, the values given as (c) are minimal estimates).

two parents, both phenotypes are produced in essentially all the mixedly infected bacteria. Even with unequal inputs of h2+ and h4+ parents most bacteria produce phage of both phenotypes. Phenotypic association of host range and serological specijicity. It has been shown that the h2+ and the h4+ host ranges were determined by allelic factors, which determine also the T2 and T4 serotypes respectively (Streisinger, 1956). Phenotypic mixing of serotype was examined by exposing progeny particles from the cross r+h4+ X r2zh2+to anti-T2 and anti-T4 sera and measuring the fractions surviving exposure to each antiserum. Heterologously absorbed sera, which do not inactivate any heterologous phage, were used. As can be seen from Table 5, three classes of serotype, analogous to the previously described host range classes, are found: a class inactivable by anti-T2 serum only, a class inactivable by anti-T4 serum only, and a class inactivable by both sera. All classes include particles that are genetically h4f and particles that are geneticallv h2+. On the other hand, after one further cycle of growth on B under conditions of single infection the genetically h2+ and h4+ particles are inactivated only by anti-T2 and anti-T4 sera respectively. The phenotypic association of host range and serotype could be examined more critically by singling out from h2+ X h4+ crosses that class of progeny particles that has the h2+ phenotype (not adsorbable by B/2) or the class of particles with h4+ phenotype (not adsorbable by B/4), and measuring their inactivability by anti-T2 and anti-T4 sera

PHENOTYPIC

MIXING

OF

TABLE NEUTRALIZATION OF PHENOTYPICALLY h4+ X h2+ (CROSS 4) BY ANTI-T2

6

h2+ AND SERUM

h'+ P.4RTICLES ANT) BY ANTI-T4

Fractions

Class of particles

Unadsorbed IJnadsorbed

by B/4 by B/2

:39*5

BACTERIOPHhGE

Phenotype

h”f h2+

surviving

anti-T2 serum

95% 7%

FROM A CROSS SERUM

treatment

with

anti-T4 serum

10%

84%

NOTE: Serum neutralization was carried out, with absorbed sera acting onl? on homologous phage. The time of exposure to antiserum was sufficient to permit phage. inact,ivation of over 9570 of inactivable

respect,ively. As shown in Table 6, particles with h”+ phenotypes were inactivated by anti-T4 serum, but only very slightly by anti-T2 serum, while particles wit,h E’+ phenotypes were inactivat)ed by anti-T2 serum but only slight,ly wit’h anti-T4 serum. There thus seems to be a close phenotypical association of host range and serological specificit)y. DISCUSSIO?r’ The site of phenotypic mixing. Both the site of adsorption of phage to the sensitive host (Anderson, 1953) and the site of inactivation of phage by antiserum (Lanni and Lanni, 1953) are believed to be on the phage tail. The site responsible for adsorption is of protein nature, since agents capable of blocking amino groups can prevent adsorption of phage T2 (Puck and Tolmach, 1954) and protein denaturing agents such as urea can activate the adsorbability of certain strains of T4 with kinetics similar to those for protein denaturation (Sato, 1956). Phenotypic mixing involves then at least the proteins of the tip of the tail. There is no information about phenotypic mixing of the phage head, which is antigenically different from the phage tail (Lanni and Lanni, 1953). Progeny particles from crosses h2+ X W fall into three groups wit,h respect to host range phenotype: W+ like, h4+ like, and h?+ W like. Assuming that there is no interaction between the h”+ and h”+ alleles at) t,he level of the synthesis of their products, so that h*+ or h4+ protein elements respectively are produced under the control of these alleles, there must be two or more sites per particle, each of mhicah can bc> filled by an h2+ or an h4+ element.

396

GEORGE

STREISINGER

The mechanism of phenotypic mixing. The association of phenotypes with nonhomologous genotypes could result from the production of h*+ and h4+ protein elements in a pool, followed by a random incorporation of these elements into the complete phage particles during the course of maturation. Such a model is suggested by the finding that similar fractions of genetically P+ and h4+ particles are found in each of the three phenotypic classes in the progeny from h*+ and h4+ crosses. The preferential association between homologous genotype and phenotype that is observed in some crosses (cross 1, Table 1) may result from an inhomogeneity in the relative yields of the two parents from bacterium to bacterium : bacteria in which an excess of a given genotype is produced would produce an excess of the corresponding phenotype (as found by Hershey in T2 h X T2 h+ crosses, personal communication). This, in turn, would lead to an apparent preferential association of homologous genotypes and phenotypes in the mass lysate. Inhomogeneities in the spatial distribution of h2+ and h4+ elements within a bacterial cell would be expected to increase this association further. A different model, which is not excluded by the present results, would assume that the h2f or h4+ protein elements are formed during the maturation of a phage particle and remain associated with the phage particle which formed them, and which carries the homologous locus, unless they are exchanged with elements from another particle. Phenotypic mixing would result from exchange of elements between particles with different genotypes. The number of exchanges per particle would have to be rather high to account for the lack of preferential association of homologous genotype and phenotype that is observed in some crosses (see cross 4, Table 1). The exchanges could furthermore occur only during a short span of time, approximately constant,, for each particle. If exchanges could occur for a long and variable time interval, for example, among fully mature particles, a shift in the frequency of association between homologous genotype and phenotype would be expected as a function of the time of intracellular development : at early lysis times, particles with few exchanges would be selected, whereas at late lysis times the particles would have had a higher mean number of exchanges. No change in the association between homologous genotype and phenotype as a funct.ion of time was observed in several lysates. The number qf adsorption elements per phage particle. As mentioned above, the portion of the phage particle subject to phenot,ypic mixing

PHENOTYPIC

MIXING

OF

BACTERIOPHAGE

397

must include at least two sites, each of which can be functional and each of which can be occupied by either an h2+ or an h"+ element. The fact that only about half the progeny is phenot.ypically h2+h4+ could be interpreted in several ways: 1. There are few sites per part(icle. 2. There are many sites, but a relatively constant’ ratio of h2+ and h4+ sites is needed for the h2+h4+phenotype. 3. There are many sites, but owing to inhomogeneous distribution of t,he elements from bacterium to bacterium or within each bacterium there is a high probability for a particle to be built exclusively with h2+ or h4+ elements. An extreme form of bacterial inhomogeneity was excluded by demonstrating t,hab each bacterium mixedly infected with h2+ and h4+ particles liberated progeny with each of the two phenotypes. ACKBOWLEDGMEKTS The author wishes to express his appreciation to Dr. S. E. Luria for his guidance throughout the course of this work, to Dr. M. Delbriick for suggesting this problem and for making available unpublished observations and t)o Miss M. Sheek who provided the backcross phage strains. REFERENCES ASDERSON, T. F. (1953). The morphology and osmotic properties of bacteriophage systems. Cold Spring Harbor Symposia Quant. Biol. 18, 197-203. DELBR~~CK, M., and BAILEY, W. T., JR. (1946). Induced mutations in bact,erial viruses. Cold Spring Harbor Symposia Quant. Biol. 11, 33-37. DOERMANN, A. H. (1948). Lysis and lysis inhibition with Escherichia coli bacteriophage. J. Bacterial. 66, 257-276. DOERMANN, A. H. (1952). The intracellular growth of bacteriophages, I. Liberation of intracellular bacteriophage T4 by premature lysis wit,h another phage or with cyanide. J. Gen. Physiol. 36, 645656. FRENCH, R. C., GRAHAM, A. F., LESLEY, S. M., and v.4~ RIMYES, C. E. (1952). The contribution of phosphorus from T2rf bacteriophage to progeny. J. Bat,teriol. 64, 597-607. HERSHEY, A. D., and ROTMAN, R. (1949). Genetic recombination between hortrange and plaque-type mutants of bacteriophage in single bacterial cells. Genetics 34, 44-71. HERSHEY, A. I)., ROESEI,, C., CHASE, M., and FORMAN, S. (1951). Growth and inherit’ance in bacteriophage. Carnegie Inst. Uvash. Yearbook ,Vo. 50, 195-200. LANNI, F., and LANSI, Y. T. (1953). Ant,igenic structure of bacteriophage. Cold Spring Harbor Symposia Quant. Biol. 18, 159-168. ?;OVICK, .4., and SZIL4RD, L. (1951). Virus strains of identical phenot,vpe but different genotype. Science 113, 34-35.

398

GEORGE

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PUCK, T. T., and TOLMACH, L. J. (1954). The mechanism of virus attachment to host cells, IV. Physicochemical studies on virus and cell surface groups. ilrch. Biochem. and Biophys. 61,229-245. SATO, G. (1956). The effect of urea on cofactor requiring bacteriophage. Thesis, California Institute of Technology, Pasadena, California. STREISINGER, G. (1956). The genetic control of host range and serological specificity in bacteriophages T2 and T4. Virology 2, 377-287. VISCONTI, N., and DELBR~~CK, M. (1953). The mechanism of genetic recombination in phage, Genetics 38, 5-33.